The present invention relates to a method for identifying genetic instability and screening for genes involved in genetic alterations by using genes that are capable of inducing genetic instability. More specifically, the present invention provides a method for generating genetically unstable cell lines by using the human metastasin 1.
Neoplastic cells typically possess numerous lesions, which may include sequence alterations such as point mutations, small deletions, insertions and/or gross structural abnormalities in one or more chromosomes such as large-scale deletions, rearrangements, or gene amplifications (Hart and Saini, 1992 Lancet, 333:1453-61; Seshadri et al., 1989 Int. J. Cancer, 43:270-72. Based upon this general observation, it has been suggested that cancer cells are genetically unstable and that acquisition of genomic instability may represent an early step in the process of carcinogenesis and a general feature of many human tumors (Liotta et al., 1991 Cell, 64:327-36. The ensuing genetic instability drives tumor progression by generating mutations in oncogenes and tumor-suppressor genes, which leads to the clonal outgrowth of a tumor (Ponta et al., 1994 BBA, 1198:1-10; Berstein and Liotta, 1994 Current Opt. In Oncology; 6:106-13; Brattein et al., 1994 Current Opt. In Oncology; 6:477-81; Fidler and Ellis, 1994 Cell, 79:315-28). These mutant genes provide cancer cells with a selective growth advantage by promoting resistance to immune-based destruction, allowing disobeyance of cell cycle checkpoints that would normally induce apoptosis, facilitating growth factor/hormone-independent cell survival, supporting anchorage-independent survival metastasis, reducing dependence on oxygen and nutrients, and conferring resistance to cytotoxic anticancer drugs and radiation. Therefore, elucidation of the genes that cause genetic instability in cancer cells, as well as identification of the genes that are most susceptible to the alterations, represents a promising approach for development of new strategies for combating cancer and for its diagnosis at early stages of development.
A. Genomic Instability
Genomic instability in its broadest sense is a feature of virtually all neoplastic cells. In addition to the mutations and/or gene amplification that appear to be a prerequisite for the acquisition of a neoplastic phenotype, human cancers exhibit other markers of genomic instability, and in particular, a high degree of aneuploidy. Many studies have shown that aneuploidy is an almost invariant feature of cancer cells, and it has been argued by some that the emergence of aneuploid cells is necessary step during tumorigenesis. The functional link between genomic instability and cancer is strengthened by the existence of several xe2x80x9cgenetic instabilityxe2x80x9d disorders of humans that are associated with a moderate to severe increase in the incidence of cancers. These disorders include ataxia telangiectasia (Gonzalez-del Angel, A. et al., 2000 Am. J. Med. Genet., 90:252-4), Bloom""s syndrome (Karow, J. K. et al., 2000 Proc. Natl. Acad. Sci. USA, 97:6504-8), Faconi anemia (Leteutre, F. et al., 1999 Brit. J. Hematol., 105: 883-93), xeroderma pigmentosum, and Nijmegen breakage syndrome (Mathur, R. et al., 2000 Indian Pediatr. 37: 615-25), of all which are very rare and are inherited in a recessive manner. Analysis of the cells from such cancer prone individuals is clearly a potentially fruitful approach for delineating the genetic basis for instability in the genome. It is assumed that by identifying the underlying cause of genetic instability in these disorders, one can derive valuable information not only about the basis of particular genetic diseases, but also about the underlying causes of genomic instability in sporadic cancers in the general population.
Currently, methods and strategies for identifying the pathology of related genes include genetic analysis of hereditary diseases, cytological methods including analysis of chromosomal aberrations and segregation analysis, and molecular biology methods including comparative genomic hybridization, microsatellite analysis, differential screening, differential display, methods of gene fishing, transgenics, knockout animals and gene function analysis.
Genomic instability plays a leading role in tumor progression and formation of metastatic cancer. This process involves activation of oncogenes, rearrangement of chromosomes, karyotipic, genetic and epigenetic instability and amplification of genes (Hart and Saini, 1992 Lancet, 339:1453-61; Seshadri et al., 1989 Int. J. Cancer, 43:270-72). The metastatic process depends not only on transformation, but also on a chain of interactions between tumor cells with the host""s cells and tissues (Ponta et al., 1994 BBA, 1198: 1-10; Berstein and Liotta, 1994 Current Opt. In Oncolgy, 6:106-13; Brattein et al., 1994 Current Opt. In Oncolgy, 6:77-81; Fidler and Ellis, 1994 Cell, 79:315-28). A series of experiments revealed that neoplastic transformation resulted in changes in expression of genes coding Ca++ binding proteins from the S100 gene family (Ebralidze et al., 1989 Gen. Dev., 3:1086-93; Schafer and Heizmann, 1996 TIBS, 21:134-40). Expression of genes encoding metastasin (S100A4) (Ebralidze et al., 1989 Gen. Dev., 3:1086-93), calcycline (S100A6) (Tomasetto et al., 1995 Genomics, 28:367-76), psoreasine (S100A7) and S100C (Moog-Lutz et al., 1995 Int. J. Cancer, 63:297-03) was enhanced, whereas expression of gene S100A2 was weakened (Lee et al., 1992 Proc. Nat. Acad. Sci. USA, 89:2504-08). It is hypothesized that these proteins participate in the tumor progression and metastasis through regulation of cell cycle and differentiation (Schafer and Heizman, 1996 TIBS, 21:134-40). The human protein metastasin-1, (hereinafter, xe2x80x9cmts-1xe2x80x9d), which is also referred to as its rodent homologue known also under the names of S100A4, calvasculin, cap1, p9ka, 42A, 18A2 and pEL98 as well the rodent homolog of the human protein has attracted attention from cancer researchers. The expression of mts-1 is observed in various aggressive cell strains (Baraclough et al., 1987 J. Mol. Biol., 29:293-98; De Vouge and Mukerjee, 1992 Oncogene, 7:109-19). Moreover, it has been demonstrated that transfection of malignant rodent cell strains with the gene metastasin may enhance metastasis. (Grigorian et al., 1993 Gene, 135:229-38 and Davies et al., 1993 Oncogene, 8:999-1008). For example, when mouse embryonic fibroblasts were transfected with the scr gene, the rat homologue of mts-1, the protein pEL98 was co-localized on cytoskeleton elements identical with actin filaments and interacted with the heavy chain of non-muscular myosin.
Chromosome instability is a characteristic cytogenetic feature of a number of genetically determined disorders collectively referred to as the chromosome breakage syndrome or DNA repair disorders. These disorders are characterized by their increased susceptibility and frequency to chromosomal breakage and chromosome interchanges occurring either spontaneously or following exposure to various DNA damaging agents. These genetic disorders share a number of features. They are all autosomal recessive, demonstrate an increased tendency for chromosomal aberrations and development of malignancies. The principal diseases in this group, which have diverse etiologies and clinical manifestations, include Fanconi anemia (FA), ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), Bloom syndrome (BS) (Karow, J. K. et al, Proc Natl Acad. Sci. USA 2000; 97: 6504-8), xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD). The underlying defect in each of these syndromes is the inability to repair a particular type of DNA damage. A number of phenotypes are caused by more than one gene. The initial diagnosis of these syndromes is made by the characteristic clinical features specific to each disease, but the definitive diagnosis is achieved by laboratory investigations such as cytogenetic, biochemical and molecular methods.
B. Genetic Diseases
Multiple genetic alterations are commonly observed in human cancers and other genetic diseases as described below.
ICF Syndrome
ICF syndrome, characterized by immunodeficiency, centromeric region instability and facial anomalies is a unique DNA methylation deficiency disease diagnosed by chromosomal anomalies, especially in the vicinity of the centromeres of chromosomes 1 and 16 (Chr 1 and Chr 16) in mitogen-stimulated lymphocytes. These aberrations include decondensation of centromere-adjacent heterochromatin, (qh) multiradial chromosomes with up to 12 arms, and whole-arm deletions.
Bloom""s Syndrome
Bloom""s syndrome (BS) is a rare human autosomal recessive disorder characterized by an increased risk of developing all types of cancer. Cells of BS patients are characterized by a generalized genetic instability including a high level of sister chromatid exchanges. BS arises through mutations in both alleles of the BLM gene, which encodes a 3xe2x80x2xe2x86x925xe2x80x2 DNA helicase identified as a member of the RecQ family.
Werner Syndrome
Werner Syndrome (WS) is an autosomal recessive disease characterized by early onset of many features of aging, by an unusual spectrum of cancers and by genomic instability. The WS protein (WRN) possesses 3xe2x80x2xe2x86x925xe2x80x2 DNA helicase and associated ATPase activities, as well as 3xe2x80x2xe2x86x925xe2x80x2 DNA exonuclease activity.
Rothmund-Thomson Syndrome
Rothmund-Thomson Syndrome (RTS) is described in three isolated patients whose main features are bilateral radial aplasia, short stature, an inflammatory based xe2x80x98elasticxe2x80x99 pyloric stenosis, a pan-enteric inflammatory gut disorder that appears to be due to an autoimmune process, and poikiloderma. Other features in individual cases include cleft palate, micrognathia, and atresia, patellar aplasia/hypoplasia and sensorineural deafness. This combination may represent a severe form of RTS or possibly a previously unrecognized condition. Two kinds of RTS were recently shown to segregate for mutations in the human RECQL4 helicase gene.
Lixe2x80x94Fraumeni Syndrome
Lixe2x80x94Fraumeni Syndrome (LFS) is a rare familial multi-cancer syndrome characterized by the occurrence of sarcomas, breast cancer, brain tumors, leukemia, and adrenal cortical tumors in multiple relatives. In most cases, LPS is the result of inheritance of a mutant TP53 followed by somatic loss of the remaining wild-type allele, which constitutes the primary initiating event leading to cancer. (Sodha, N. et al., 2000 Science, 289: 359).
Fanconi Anemia
Fanconi Anemia (FA) is an autosomal genetic disease characterized by a complex array of developmental disorders, a high predisposition to bone marrow failure and to acute myelogenous leukemia. The chromosomal instability and hypersensitivity to DNA cross-linking agents led to its classification as a DNA repair disorder. (de Winter, J. P. et al., 1998 JOURNAL 8: 281-3). de Winter questioned whether it was appropriate to consider 1/approximately FA within a DNA repair framework in view of the recently discovered genetic heterogeneity characteristics of the defect (eight complementation groups). One possibility is that the FA proteins interact to form a complex which may control different functions, including the processing of specific DNA lesions. Such a complex may act as a sensor to initiate protective systems as well as transcription of specific genes specifying, among other proteins, growth factors. Such steps, may be organized as a linear cascade or more likely under the form of a web network.
Nijmegen Breakage Syndrome
Nijmegen Breakage Syndrome (NBS) is characterized by extreme radiation sensitivity, chromosomal instability and cancer. The phenotypes are similar to those of ataxia telangiectasia mutated (ATM) disease (Gonzalez-del Angel A., et al., 2000 Am. J. Med. Genet. 90: 252-4), in which there is a deficiency in a protein kinase that is activated by DNA damage, indicating that the Nbs and Atm proteins may participate in common pathways. Nbs is specifically phosphorylated in response to gamma radiation, ultraviolet light and exposure to hydroxyurea. Phosphorylation of Nbs mediated by gamma radiation, but not that induced by hydroxyurea or ultraviolet light was markedly reduced in ATM cells. In vivo, Nbs was phosphorylated on many serine residues of which S343, S397 and S615 were phosphorylated by Atm in vitro. At least two of these sites were underphosphorylated in ATM cells. Inactivation of these serines by mutation partially abrogated Atm-dependent phosphorylation. Reconstituting NBS cells with a mutant form of Nbs that cannot be phosphorylated at selected ATM-dependent serine residues led to a specific reduction in clonogenic survival after gamma radiation. Thus, phosphorylation of Nbs by Atm is critical for certain responses of human cells to DNA damage.
Ataxia-telangiectasia
The human neurodegenerative and cancer predisposition condition ataxia-telangiectasia (AT) is characterized at the cellular level by radiosensitivity, chromosomal instability, and impaired induction of ionizing radiation-induced cell cycle checkpoint controls. Recent work revealed that the gene defective in ataxia-telangiectasia, termed ATM, encodes an approximately 350 kilodalton (kDa) polypeptide. ATM is a member of the phosphatidylinositol 3-kinase family.
Cockayne Syndrome
Cockayne Syndrome (CS) is a human autosomal recessive disorder characterized by many neurological and developmental abnormalities. CS cells are defective in the transcription coupled repair (TCR) pathway that removes DNA damage from the transcribed strand of active genes. Individuals suffering from CS do not generally develop cancer but show increased neurodegeneration. (Senesen, M. et al., 2000 Nucleic Acids Res. 28: 3151-9).
Trichothiodystrophy
Trichothiodystrophy is an autosomal recessive genodermatosis associating congenital dysplasia of the hair and neuroectodermal defects. Clinical expression is variable, although abnormalities are generally noted from birth. The DNA repair-deficient genetic disorders, xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) result from mutations in the XPD gene with different sites of mutation. Characteristics of XP are multiple pigmentation changes in the skin and a greatly elevated frequency of skin cancers. XPD and most TTD patients have reduced levels of DNA repair, but some reports have suggested that the repair deficiencies in TDD cells are milder than in XP-D cells.
The present invention describes a method for understanding the role of genomic instability in tumor progression and formation of metastatic cancer by identifying unstable genes associated with genetic instability. More specifically, the present invention involves a method for detecting genes associated in genetic alterations by using specific genes capable of inducing genetic instability such as mts-1 and their protein products.
The present inventors discovered that cells that were transfected with mts-1 and later expressed mts-1, resulted in acquired genomic instability. Based on this finding, the present invention provides a method for using the human mts-1 gene or its protein product, or their analogs, as well as other genes, their protein products and their analogs that have a similar function to that of mts-1 gene, to induce genetic instability in tissues and cell lines. Therefore, the present invention provides a method for detecting unstable genes by comparing the phenotype and gene expression pattern of subclones that express mts-1.
The present invention also provides a method for diagnosing and identifying genetic elements that are involved in a pathological process, as well as a tool for facilitating genome instability research. In addition, the present invention further provides a method for using these genetically induced unstable cell lines in kits for analysis of genome instability by genome dose variability array (GDVA).
The present invention provides a method for identifying genes that are involved in genetic instability comprising the steps of transfecting cells with a vector expressing a gene capable of inducing genetic instability; selecting the cells that express the incorporated gene capable of inducing genetic instability; subcloning the cells; comparing the phenotypes of the subclones that express the incorporated gene capable of inducing genetic instability; and comparing the pattern of genetic expressions of the subclones. The present invention demonstrates that the mts-1 gene and its analogs and their protein products can be used to induce genome instability in stable cells and tissues.
Human breast carcinoma MCF7 cells were stably transfected with a vector expressing human mts-1. More importantly, the present invention demonstrates that genomic instability is induced by cells that express the mts-1 gene. In particular, selected MCF7 cell clones expressing mts-1 showed a remarkable variation in their phenotype in terms of their morphology, proliferation rate, sensitivity to estradiol and pattern of gene expression. Among the obtained cell clones, 40% demonstrated multilayer cell growth, indicating immortalization. After re-cloning, these cell strains showed a continued diversification of cell clones with respect to their phenotypes.
In a preferred embodiment, cells are transfected with a vector containing the human metastasin (mst-1) gene and its analogs and their protein products. Cells are selected for expression of mst-1 and subcloned to several generations and analyzed for diversity in phenotype and genetic expression patterns.
It was noted that in the subclones expressing mts-1, the proviral genome IAP (HERV-67) was eliminated whereas the pS2-gene was amplified. In addition, the pS2 and p53 genes, respectively showed a loss of correlation in a similar manner when compared to the dose of certain genes (i.e., uPA, TIMP-1, IAP and Bcl-x), whereas subclones of parental MCF7 cells and MOCK-transfected MCF7 control cells demonstrated a good internal correlation at comparisons between all genes. The loss of correlation between dose of different genes in mts-1-expressing cells indicates that mts-1 causes an unstable genome, which might explain the observed progressive diversification of phenotypes.
In another embodiment, patterns of genetic expression are analyzed using a known marker gene such as but not limited to the proviral genome IAP (HERV-67), uPA, TIMP-1, IAP, Bcl-x, p53 and pS2. Genomic DNA samples are isolated from clones, subclones, subpopulations of cells, solid tumors or from subpopulation of non-solid tumors in which genomic instability is induced by mts-1 gene or by its functional analogs. These samples may be labeled with dye or radioisotope, or any other tag known to one skilled in the art. The labeled samples are hybridized with different sets of cDNA, mRNA, oligonucleotide probes or non-coding DNA and RNA probes that are immobilized to an array. Commercial arrays can be used without limitation, such as Human Broad Coverage cDNA Microarray; Human Apoptosis; Human Cancer; Human Cell Cycle; Human Cell Interaction; Human Cytokine/Receptor; Human Hematology; Human Neurobiology; Human Oncogene/Tumor Suppressor; Human Stress; Human Cardiovascular; Human Trial Kit; Human Cancer 1.2; xcex2-Kinase; xcex2-Signal Transduction; xcex2 Metabolism; xcex2 Diagnostic expressed sequence tags; Human 1.2 I ; Human 1.2 II; Human Toxicology II; Human 1.2 III; Human Functional cDNA Microarray; Atlas Glass Human 1.0 Array; Mouse cDNA Broad Coverage MicroArray; Mouse Stress; Mouse 1.2; Mouse 1.2 II; Mouse 1.2 Cancer; Rat Stress; Rat 1.2; Rat 1.2 II; Rat Toxicology II; E. coli Gene Arrays; H. pylori Gene Arrays; B. subtilis Gene Arrays; H. Pylori ORFmers sets; B. subtilis ORFmer sets; E. coli ORFmer sets.
Further, the probe sets could be assembled using various sources such as, without limitation, cancer related genes, retroviral DNA or RNA, DNA fragments, cDNA and RNA extracted from various viruses, microorganisms, plants, insects and animals, synthetic oligonucleotides and their analogs or mimics. Analysis of instability is performed by calculating correlation coefficients (r) for each couple of probes, or by other statistical methods as described below.
In another embodiment, the genomic DNA samples are immobilized to an array and hybridized with sets of cDNA, mRNA, oligonucleotide probes, as well as non-coding DNA and RNA probes that are labeled with a dye or isotope, or any other tag. The probe sets could be assembled using various sources such as, without limitation, cancer related genes, retroviral DNA or RNA, DNA fragments, cDNA and RNA extracted from various viruses, microorganisms, plants, insects and other animals, synthetic oligonucleotides and their analogs and mimics. Analysis of instability is done by calculating correlation coefficients (r) for each couple or by other statistical methods as described below.
Analysis of instability can be performed by using statistical methods, for example, by calculating correlation coefficient (r) for each couple of genomic DNA samples. The following correlation analysis methods can be used without limitation: multiple regression; cluster analysis; factor analysis; classification trees; canonical analysis; multidimensional scaling; correspondence analysis; linear regression analysis.
In still another embodiment, analysis of genetic instability in the samples is performed by using comparative genomic hybridization; microsatellite analysis; differential display analysis; or any other similar methods that allow identification of genetic alterations.
In yet another embodiment, analysis of genetic instability of tumor tissues or other pathological tissues is preformed by comparing patterns of gene expression using two or more known marker genes. These marker genes may be identified by employing the method of this invention. Therefore, another embodiment of the present invention is a method for identifying unstable genes in cells that are involved in genomic instability comprising transfecting cells with a vector expressing human mts-1 or its functional analogs; selecting for the cells that express mts-1; subcloning the cells that express mts-1; comparing the phenotype of the subclones that express mts-1, comparing the pattern of gene expression of subclones with two or more marker genes; and identifying the cells as possessing unstable genes if the phenotype of the subclones that express mst-1 continues to demonstrate diversity and the pattern of gene expression for the subclones that express mst-1 show a loss of correlation with the marker gene when compared to other subclones that express mst-1. Genomic DNA, mRNA or cDNA isolated from samples obtained from the analyzed tissue samples are immobilized on arrays and hybridized with the marker genes that are labeled with radioactive or fluorescent probes or any other suitable tag. The correlation analysis between the doses of the marker genes in the samples is performed as described above. Such an analysis can be used without limitation for genetic diagnostics of the disease, evaluation of its development stage and its progression prognosis.
The results of the analysis generated from the present method can be used for evaluation of genetic instability of new genetically altered organisms and cell lines. In another embodiment a single gene or groups of genes that are identified by the above-described strategies, or their expression products are used as targets for therapeutic agents or for preparation of therapeutic and preventive vaccines against the disease.
In still another embodiment, genetic instability is stimulated ex vivo by using unstable genes of the present invention in tissue samples obtained from an individual patient to promote genetic alterations that are characteristics for disease progression in this particular individual. The single antigens or groups of antigens, or genetic material such as mRNA or cDNA, are further used to prepare therapeutic and preventive vaccines against the disease.
In another embodiment, the present invention can be used for diagnostics and identification of genetic elements involved in a pathological process related to genome instability.